The first squalene synthase (SS) gene to be functionally characterized was isolated from Saccharomyces cerevisiae and cloned concurrently by the Karst and Robinson groups (1,2). Both groups utilized the strategy of screening S. cerevisiae genomic library clones for their ability to functionally complement a squalene synthase (erg9)-deficient yeast line. Interestingly, Jennings, et al. (2) found that a genomic clone containing only a partial SS gene fragment was able to restore ergosterol prototrophy even though it only restored 5% of the normal level of SS enzyme activity. This finding suggested that low levels of SS enzyme activity were sufficient to complement the erg9 deficiency in yeast. Soon afterwards, Robinson, et al. (3) attempted to clone the SS gene from Homo sapiens and S. pombe using the same strategy, but isolated only the S. pombe gene by screening for complementation of the er9-deficient line (3). Having two SS genes from two species of fungi, these investigators were able to identify conserved regions within the deduced protein sequences to which they designed degenerate primers and cloned the human SS homolog using PCR (3). Robinson, et al. (3) confirmed that the human squalene synthase gene was unable to restore ergosterol prototrophy to the erg9-deficient yeast line, but a chimera SS gene constructed by combining a 5′ region of the human gene containing the putative catalytic domain with a 3′ region of the S. cerevisiae gene containing a membrane-anchoring domain was able to complement the erg9 deficiency. Robinson, et al. (3) suggested that the inability of human SS to functionally complement the erg9-deficient yeast line was due to problems with expression or stability of the human protein in S. cerevisiae. A few years later, Soltis, et al. (4) isolated a similar allele of the human squalene synthase gene by screening a human cDNA library with a rat squalene synthase gene probe. These investigators also determined that the human squalene synthase gene was not able to complement an erg9 deficiency in yeast. They were, however, able to document expression of the human squalene synthase gene in yeast by recording the corresponding protein by immuno-blotting methodology, as well as measuring inducible enhancement of SS enzyme activity. This result conflicted with the notion that a heterologously expressed SS was not able to complement the erg9 deficiency in yeast because of problems with transgene expression or protein stability in yeast, and Soltis, et al. (4) hypothesized that structural differences in the carboxy-termini of the yeast and human SS may affect localization or folding of the proteins in association with intracellular membranes.
The first plant SS was cloned from Arabidopsis (5) and soon after from Nicotiana benthamiana (6). Nakashima, et al. (5) failed to isolate an Arabidopsis SS gene by screening for complementation of an erg9 deficient yeast line, and instead screened plaques of an Arabidopsis cDNA library with a mouse squalene synthase cDNA probe. Hanley, et al. (6) used a degenerate primer/PCR approach to isolate a N. benthamiana SS, and likewise noted that the tobacco SS gene was unable to restore growth when expressed in an erg9 deficient yeast strain. Later, Kribii et al. (7) reported that the Arabidopsis genome contained two highly homologous SS genes organized in a tandem array. This group confirmed that the Arabidopsis SS could not complement the erg9 (SS gene) disruption in yeast, but they measured significant SS enzyme activity in the microsomal fraction of these yeast. These investigators went on to show that a chimeric Arabidopsis SS gene containing a substitution corresponding to the 66 carboxy-terminal amino acids of Arabidopsis SS with 111 carboxy-terminal amino acids of the S. pombe SS were sufficient to restore prototrophic growth of the erg9 knockout in yeast without exogenous sterol. Radiolabeling studies were also performed with [3H]-FPP fed to microsomes isolated from yeast expressing either the full length Arabidopsis SS or the Arabidopsis-S. pombe chimera SS genes, or from wild type yeast. Radiolabel was incorporated by either the wild type yeast microsomes or microsomes from the erg9-deficient yeast over-expressing the Arabidopsis-S. pombe chimera SS into squalene, squalene-2,3-epoxide, and lanosterol. However, when [3H]-FPP was incubated with microsomes from erg9 deficient yeast expressing the full length Arabidopsis SS, only radiolabeled squalene was detected. No SS enzyme activity was detectable in the cytosolic (soluble) fractions of these yeast lines. These results strongly suggested that active SS was being expressed and targeted to membrane in all the constructs tested; however, the carboxy-terminal 111 amino acids of S. pombe were necessary for channeling of squalene into the ergosterol biosynthetic pathway (7).
In 2000, another fungal squalene synthase was isolated from Yarrowia lipolytica using a degenerate primer approach (8). The Y. lipolytica SS was found to complement an erg9 deficient yeast line, albeit the complemented yeast grew slower than the yeast complemented with the S. cerevisiae SS gene. Altogether, this result and those of the other investigators demonstrated that at least three different fungal SS could complement the erg9 knockout in S. cerevisiae, but no other SS isolated from animal or plant could accomplish this task.
In 2008, Busquets, et al. (9) reported that of the two annotated SS genomic sequences in Arabidopsis, only one coded for a functional SS enzyme. Busquets, et al. also performed some fluorescence microscopy experiments to determine the intracellular location of Arabidopsis SS (9). GFP was tagged to the N-terminus of a full length SS, a SS lacking the equivalent of the carboxy-terminal 67 amino acids, or the GFP was fused directly to a gene fragment corresponding to that encoding for the carboxy-terminal 67 amino acids of the SS. All three constructs were transiently co-expressed in onion epidermal cells with an ER-targeted version of DsRed. Both the GFP linked to the full length SS and the carboxy-terminal 67 amino acids of SS co-localized with DsRed, which indicated that these two SS enzymes were localized to the ER membrane. The GFP-SS fusion lacking the carboxy-terminal 67 amino acids appeared localized to only the cytosol. These authors concluded that the membrane-spanning region at the carboxy-terminus of SS was critical for correct targeting of SS to the ER membrane (9).
These results and the present inventors' observations that the algal Botryococcus braunii SS also could not complement the erg9 mutant in yeast suggested that it was not simply targeting of squalene synthase enzyme activity to the ER membrane of yeast that was important. Some additional protein domain within the carboxy-terminal region of the yeast squalene synthase was necessary to facilitate the complementation phenotype.